1. Field of the Invention
The present invention relates to a method for long-term culturing of avian spermatogonial stem cells, a population of avian spermatogonial stem cells and a method for producing transgenic aves.
2. Description of the Related Art
Spermatogenesis is a process involving division and differentiation of spermatogonial cells in testis of male animals and apoptosis of cells. Therefore, the spermatogenesis is very complex, systematic and effective process. The spermatogenesis in chicken is very similar to that of mammals, involving the complicated interaction between seminiferous tubule and interstitial cells.
Spermatogonial cells of avians originate from primordial germ cells (PGCs) that are derived from the epiblast and gradually move to the lower layer during the early stages of primitive streak formation. PGCs then translocate to the hypoblast and colonize at the germinal crescent. They circulate into the developing blood vascular system and migrate to the germinal ridge, finally differentiating into spermatogina in testis.
Spermatogonial cells have capacities of self-renewal and spermatogenesis (Morrison et al., 1997). In mice, a spermatogonial cell becomes spermatocyte through about ten times divisions. That is to say, a stem cell becomes 1024 spermatocytes and then 4096 spermatozoa following a series of meiosis. 75-95% of spermatozoa generated disappear by apoptosis.
Testes have a lower population of spermatogonial stem cells. For example, it has been suggested that 2×104 stem cells exist in a mouse testis having approximately 108 cells (Meistrich & Beek, 1993; Tegelenbosch & de Rooij, 1993). A spermatogonial stem cell has become highlighted among spermatogonia because of its self-renewing and spermatogenesis potentials throughout adult life span.
Various attempts have been made to reproduce in vitro spermatogenesis using isolated germ cells; however, those have been finally unsuccessful. Rassoulzadegan et al., 1993 have reported that immature germ cells of rat are co-cultured with Sertoli cells to differentiate into haploid spermatid. However, there remain technical limitations in in vitro spermatogenesis. Hitherto, in vitro culture systems for spermatogonia have been reported to be practical only within several weeks (Ogawa, 2001; Dirmai et al., 1999; Nagano et al., 1998). It has been reported that spermatogonial cells were cultured for about 4 months and then introduced into a recipient to give rise to normal spermatogenesis (Nagano et al., 1998). The culturing of spermatogonial cells remains difficult because spermatogonial cells are isolated in a restrictive manner and its higher proportion dies during culture. In particular, morphological and biochemical markers to discriminate spermatogonial stem cells from spermatogonial cells differentiated have not yet been suggested, which is considered the greatest obstacle (Nagano et al., 1998; van Pelt et al., 2002).
Shinohara et al. (1999) have reported that antibodies against α6-integrin and β1-integrin show reactivity to spermatogonial stem cells from mice different from other tissue cells, demonstrating that they may serve as markers. DBA (Dolichos biflorus agglutinin) exhibits a specific reaction pattern for 30 weeks after birth to gonocyte and spermatogonia in bovine testis, ensuring the lectin may serve as markers (Ertl and Wrobel, 1992).
For mammals such as rat, spermatogonial cell line established by culturing has not been reported. Instead, there have a few reports in which spermatogonial cell lines of rat and mouse may be established using mTERT (mouse telomerase catalytic component) (Feng et al., 2002) or SV40 large T antigen (van Pelt et al., 2002).
Dobrinski et al. (2000) have cultured testicular cells from livestock such as cattle, pig and horse and then introduced cells into mouse testis. Izadyar et al. (2003) have studied the division and differentiation patterns of spermatogonial cells during long-term (about 150 days) culturing of bovine type A spermatogonial cells. The culture of spermatogonial cells from human has been performed mainly for treating diseases or disorders such as azospermia; however, where cells differentiated to spermatid were used for fertilization, it was observed that they did not develop to morula and lead to sex chromosome aberration (Sousa et al., 2002).
Meanwhile, avian spermatogonial cells become highlighted as a potential tool for producing transgenic avians; however, the culture and use of spermatogonial cells from avians such as chicken have not yet been researched. Such spermatogonial cells are expected to provide a tool to elucidate molecular mechanism of spermatogenesis and also to be useful in the production of transgenic animals and gene therapy of germ cells.
Throughout this application, various publications are referenced and citations are provided in parentheses. The disclosure of these publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.